丙肝抗原决定簇与CTB的融合、表达及融合蛋白的纯化

通过固相化学合成法合成了编码丙型肝炎病毒(HCV)结构区和非结构区的4个抗原决定簇基因,这些抗原决定簇基因片段以不同方式串联后与ctxB基因融合,构建了12种表达不同嵌合蛋白的重组质粒,各重组质粒转化大肠杆菌后均能高效分泌性表达融合蛋白,表达产量在10～50μg/ml之间,随所融合的抗原决定簇不同而不同,表达水平主要与抗原决定簇的氨基酸组成有关,而与抗原决定簇的大小及串联次数关系不大。融合蛋白通过亲和层析纯化,达到了电泳纯,为进一步研究融合蛋白的抗原性及用作抗-HCV ELISA诊断试剂抗原打下了基础。     

四价HCV聚合抗原的克隆,表达及在抗—HCV ELISA试剂中的应用

本研究通过反转录从丙肝病人血清中克隆了丙型肝炎病毒（ＨＣＶ）Ｃ３３ｃ基因,通过ＤＮＡ合成法又分别合成了编码ＨＣＶ核心抗原基因,ＮＳ４抗原及ＮＳ５抗原部分抗原决定簇的基因,利用基因工程手段,在大肠杆菌中表达了含有上述四种抗原的融合蛋白。该融合蛋白具有良好的抗原性和特异性,以该融合蛋白为抗原制备的抗－ＨＣＶＥＬＩＳ试剂检测了中国药品生物制品检定所１９８份标准血清时,其阳性,阴性符合分别达９７％和９８％     

丙型肝炎病毒抗原决定簇与霍乱毒素B亚基的融合表达以及融合蛋白的抗原性

通过固相化学合成法,合成了编码丙型肝炎病毒（ＨＣＶ）结构区和非结构区的４个抗原决定簇基因,这些抗原决定簇基因片段以不同方式串朕后与ｃｔｘＢ基因融合,构建了１２种表达不同嵌合蛋白的重组质粒。各重组质粒转化大肠杆菌后均能高效分泌性表达融合蛋白,表达产量随所融合的抗原决定簇不同在１０～５０μｇ／ｍｌ之间,表达水平主要与抗原决定簇的氨基酸组成有关,而与抗原决定簇的大小及串联次数关系不大。融合蛋白通过亲和层析纯化达到了电泳纯。对１１种融合蛋白中ＨＣＶ抗原决定簇的反应原性的研究表明,多数融合蛋白中ＨＣＶ抗原决定簇均能与对应的ＨＣＶ抗体结合。其中以融合蛋白９５０８２为抗原研制的抗－ＨＣＶＥＬＩＳＡ试剂具有良好的灵敏度和特异性。     

丙型肝炎病毒不同标志物检测结果的相关性分析

目的分析丙型肝炎病毒(Hepatitis C virus,HCV)抗原、抗体及核酸标志物实验室检测结果之间的关联性,评价适合血源筛查与早期临床确诊联合检测HCV感染的实验室诊断技术。方法用HCV RNA定量试剂、HCV核心抗原试剂及HCV抗体试剂分别检测304份血浆样本中HCV RNA载量、HCV Ag和抗-HCV指标,并对HCV RNA阳性样本进行基因分型。结果在304份血浆样本中,检出HCV RNA阳性样本87份,其HCV RNA载量≥500 IU/m L、500~30 IU/m L之间和<30 IU/m L时,血清学标志物HCV Ag浓度≥3 fmol/L及抗-HCV信号值/判断值≥1的阳性率分别为92.0%(23/25):96.0%(24/25)、58.8%(10/17):82.3%(14/17)及11.1%(5/45):75.6%(34/45),HCV RNA载量低于基因分型试剂盒LOD要求为500 IU/m L时,基因分型检测率为24.2%(15/62)。HCV RNA阴性样本217份中,HCV Ag浓度≥3 fmol/L和抗-HCV信号值/判断值≥1的样本阳性率分别为3.2%(7/217)和32.7%(71/217)。血清学指标在不同HCV RNA载量的阳性率差异具有统计学意义(χ2=197.4,P<0.01),HCV RNA载量与HCV Ag和抗-HCV水平成正相关。结论在中国人群中存在低HCV RNA水平携带者,HCV Ag和基因分型检测能力需要进一步提高。     

丙型肝炎病毒核壳蛋白抗原的化学合成及其在血清学诊断中的应用

选取丙型肝炎病毒(HCV)核壳蛋白区两个可能含有优势抗原表位的19和16氨基酸的肽殴(C-19肽和C-16肽),利用固相多肽合成技术进行了化学合成。经用反相高压液相层析(HPLC)及脉冲液相多肽测序技术检定合成肽产品的纯度及序列后,以C-19和C-16肽作为包被抗原组装成检测HCV血清抗体的ELISA诊断试剂。用所组装的合成肽试剂检测中国药品生物制品检定所提供的HCV标准参比血清,并比较利用该试剂和美国Abbott实验室生产的HCV第二代EIA诊断试剂平行检测109份献血员血清的结果,表明C-19肽能够特异、重复、灵敏地检出HCV血清抗体,可以作为我国第一代HCV诊断试剂的合成肽抗原。     

HCV E2区基因在原核系统中的表达

用提取的重组表达载体pET-E2转化BL21(DE3)感受态细胞,经IPTG诱导,再进行SDS-PAGE,可得到有一条约34 kDa的表达带,与理论推测的蛋白分子量一致,通过Western-blot鉴定,证明此带即为目的蛋白带.该产物有一个六聚组氨酸尾,主要以包涵体形式存在;计算机扫描分析考马斯亮兰染色后的蛋白胶显示目的蛋白占整个菌体蛋白的36%以上,经Ni-柱纯化的E2蛋白纯度可达95%以上;以纯化的E2蛋白为抗原,用ELISA方法检测了20份抗HCV阴阳性血清,结果表明15份抗HCV阳性血清中检出5份E2抗体阳性血清,而5份抗HCV阴性血清中没有检测到E2抗体.     

HCV核心蛋白在大肠杆菌中的表达及免疫学特性分析

目的:表达HCV核心蛋白,为检测丙肝病毒提供合适抗原。方法:以含HCV核心全长cDNA克隆的pMD18T/core质粒为模板,PCR扩增全长的HCV核心抗原基因,插入表达载体pQEN1构建重组质粒pQEN1/Core,转化BL-21(DE3)大肠杆菌,IPTG诱导表达6×His融合蛋白,表达产物经SDS-PAGE及Western blot检测和鉴定。结果:经SDS-PAGE及Western blot显示HCV核心蛋白在大肠杆菌中正确表达,融合蛋白分子量约为22 kD,表达量约占菌体蛋白总量的30%。纯化后的C蛋白能与慢性丙型肝炎患者有血清反应。结论:HCV核心蛋白在大肠杆菌中成功表达并具有较强的抗原性。     

HCV E2区基因在原核系统中的表达

用提取的重组表达载体pET-E2转化BL21（DE3）感受态细胞,经IPTG诱导,再进行SDS－PAGE,可得到有一条约34kDa的表达带,与理论推测的蛋白分子量一致,通过Western-blot鉴定,证明此带即为目的蛋白带。该产物有一个六聚组氨酸尾,主要以包涵体形式存在;计算机扫描分析考马斯亮兰染色后的蛋白胶显示：目的蛋白占整个菌体蛋白的36％以上,经Ni-柱纯化的E2蛋白纯度可达95％以上;以纯化的E2蛋白为抗原,用ELISA方法检测了20份抗HCV阳性血清,结果表明15份抗HCV阳必血清中检出5份E2抗体阳性血清,而5份抗HCV阴性血清中没有检测到E2抗体。     

RT套式PCR检测血浆HCV RNA及与抗HCV检测的比较

应用微量血清热变性法提取核酸,逆转录套式聚合酶链反应(RT-nest PCR)检测血浆HCV RNA,并与抗HCV ELISA检测结果比较,对HCV RNA阳性标本进行HGV RNA的筛查.结果在32例抗HCV阳性和20例抗HCV阴性血浆中,HCV RNA分别检出18例和2例,总符合率为70%,20例HCV RNA阳性者中有2例合并感染HBV,1例合并感染HGV.证明血浆样本中抗HCV与HCV RNA间存在很大的相关性.     

四种丙型肝炎病毒抗体试剂检测方法的性能评估

对血液筛查的四种丙型肝炎病毒(Hepatitis C virus,HCV)抗体试剂检测方法的性能进行评估。方法采用协作标定方式,应用四种检测方法(间接ELISA法、双抗原夹心ELISA法、间接CLIA法、双抗原夹心CLIA法)16种HCV抗体试剂,对经确证筛选出的70份(HCV抗体阳性35份、阴性35份)血浆样本进行检测,通过分析阳性、阴性符合率,评估四种方法 HCV抗体试剂的性能。结果间接ELISA法试剂检测阳性符合率为88.6%(31/35)~94.3%(33/35),双抗原夹心ELISA法、间接CLIA法、双抗原夹心CLIA法试剂检测阳性符合率为91.4%(32/35)~94.3%(33/35),四种检测方法 HCV抗体试剂阳性符合率之间的差异无统计学意义(P0.05);间接ELISA法和间接CLIA法试剂弱阳性样本检出率为11.4%(4/35)~31.4%(11/35),双抗原夹心ELISA法和双抗原夹心CLIA法试剂弱阳性样本检出率为5.7%(2/35)~11.4%(4/35),四种检测方法 HCV抗体试剂对弱阳性样本的检出率之间的差异有统计学意义(P0.05);间接ELISA法试剂检测阴性符合率分别为94.3%(33/35)~100%(35/35),间接CLIA法试剂阴性符合率分别为94.3%(33/35)~97.1%(34/35),双抗原夹心ELISA法和双抗原夹心CLIA法试剂阴性符合率均为97.1%(34/35),四种检测方法 HCV抗体试剂阴性符合率之间的差异无统计学意义(P0.05)。结论四种检测方法 HCV抗体试剂的性能基本一致,不同方法各有优缺点,建议应用多种检测方法对弱阳性样本进行确认检测。     

A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia coli

The objective of this study was expression of a recombinant fusion protein p24-gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24-gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24-gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.     

用国产重组抗原研制丙肝病毒抗体检测试剂

用克隆表达的丙型肝炎病毒核心蛋白及非结构 3区蛋白、非结构 4区蛋白和非结构 5区蛋白作为抗原 ,研制装配了第三代丙肝病毒抗体检测试剂。用该试剂检测国家第三代丙肝病毒抗体检测试剂标准血清 ,40份阴性血清全部符合 ,40份阳性血清出现 1份假阴性。分析探讨了各种影响试剂质量的因素     

丙型肝炎病毒核心抗原高效表达及产物抗原性的研究

用含谷胱甘肽转硫酶（ＧＳＴ）基因的ｐＧｅｘ－２Ｔ为表达载体在大肠杆菌中高效表达了含ＨＣＶ核心区部分基因片段（约１２２个氢基酸）的融合蛋白,表达产物Ｃ２７经测定占菌体总蛋白的３０％,Ｃ２７纯化后,用我国ＨＣＶ诊断试剂第二代血清参考品为标准与Ｇ１１核心抗原进行比较,结果显示二者具有相同的总体检出率和近似的抗原活性。因此,推测表达的部分区段基因可能是核心区重要功能区域。另外融合蛋白对提高重组蛋白的抗原活性和分离纯化等均有一定作用。     

用细菌/杆状病毒系统在昆虫细胞中表达GFP与HCV抗原融合蛋白

用细菌／杆状病毒（Ｂａｃ－ｔｏ－Ｂａｃ）系统在昆虫细胞中高效表达了绿色荧光蛋白（ＧＦＰ）与ＨＣＶ抗原的双功能融合蛋白,经ＥＬＩＳＡ测定和荧光显微镜观查证实,表达产物既能发射易于检测的绿色荧光,又具有ＨＣＶ的抗原活性,实现了用绿色荧光蛋白等分子标记抗原,为免疫诊断新方法的建立打下了理论基础．     

A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus

African swine fever virus (ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds. Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase (nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA (cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7% by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.     

丙型肝炎病毒NS3蛋白的原核表达及多克隆抗体制备

提高对丙型肝炎患者实验室检测的灵敏度和特异性,对相关人群进行筛查和早期诊断,是控制丙型肝炎病毒(HCV)流行与传播的有效措施。为了建立更为可靠的HCV诊断方法,通过采用PCR方法从J6/JFH12a型病毒中克隆出HCV ns3基因片段,将其连接到p ET-28a载体上,重组载体p ET-28a-ns3转化大肠杆菌BL21(DE3)后诱导表达,以10%SDS-PAGE进行鉴定,获得表达的NS3重组蛋白分子量为72 k Da。将纯化的NS3蛋白免疫BALB/c小鼠,第4次免疫后采集血液并分离血清进行抗体活性鉴定,小鼠抗体效价为1∶256 000。进一步的Western blotting和间接免疫荧光结果显示,以重组NS3蛋白免疫小鼠制备的多克隆抗体可以很好地识别HCV感染Huh7.5.1细胞中的NS3蛋白,为下一步开展单克隆抗体制备和检测试剂盒研制工作奠定了基础。     

Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.     

Expression of Coat Protein of an Ordinary Strain of Potato virus Y in Escherichia coli and Production of Polyclonal Antibodies for Diagnosis

The coat protein gene (CP) of an ordinary strain of Potato virus Y (PVY^O) was cloned into the expression vector, pET‐28a(+). The insert was sequenced and analysis showed that the CP gene was in frame with intact N‐terminal 6X histidine tags. An approximately 35 kDa recombinant fusion protein was observed in inclusion bodies of induced Escherichia coli BL21 cells. This fusion protein was purified and used as antigen to raise polyclonal antibodies in rabbits. In Western blot and dot blot immuno‐binding assay (DIBA), both PVY^O‐CP IgG and PVY^O IgG strongly reacted with the recombinant CP. The PVY^O‐CP IgG could detect PVY^O in infected samples up to 1 : 3200 dilutions. A PVY^O‐CP ELISA kit was prepared and compared with conventional ELISA kit based on purified virus particles (PVY^O ELISA kit). The PVY^O‐CP ELISA kit consistently detected the PVY^O in DAS‐ELISA of field samples and was as effective as PVY^O ELISA kit.     

Expression of hepatitis B virus S gene in Pichia pastoris and application of the product for detection of anti-HBs antibody

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying a hexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5 % of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100 % with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.